Follicular helper T (Tfh) cells are a distinct subset of CD4+ helper T (Th) cells that regulate the development of antigen-specific B cell immunity. Upon exposure to a foreign antigen, Tfh cells help B cells generate antibody-producing plasma cells and long-lived memory B cells. While the specific triggers required for Th1, Th2 or Th17 polarization are well defined, those responsible for Tfh differentiation are incompletely understood. In order to identify signals that could be used to promote Tfh responses, we have utilized an experimental model in which C57BL/6 or gene KO mice are immunized with either peptide or protein antigen emulsified in the oil and surfactant-only containing adjuvant IFA (Incomplete Freunds Adjuvant). Using this immunization protocol in wild-type mice we find that at the peak of the response, half of the antigen specific CD4+ T cells have differentiated into Tfh based on their expression of the chemokine receptor CXCR5 and the lineage defining transcription factor Bcl6 while little or no increase in Th1 or Th17 cells is observed. As described in last years report, Nicolas Riteau in the lab has demonstrated that the promotion of Tfh responses by this adjuvant that lacks exogenous microbial agonists occurs through a pathway involving type I IFN and, paradoxically, the microbial sensing related adaptor molecule MyD88. Recent studies by other groups have demonstrated that the function of Tfh can be influenced by another subset of CD4+ T cells that in addition to Tfh markers expresses the regulatory cell associated master transcription factor FoxP3. During the year Dr. Riteau demonstrated that these Tfr cells are also induced in our different peptide/adjuvant immunization protocols and interestingly are generally inversely related in numbers to the Tfh induced in the same response. For example in MyD88-/- mice peptide/IFA immunization induces less Tfh but more Tfr than the corresponding response in wild-type mice. These data thus support a model where Tfh and Tfr cross regulate each others induction and activity. As introduced in last years report, we recently launched a new research project examining the influence of the gut and pulmonary microbiota on Mtb infection and vice versa . In addition, we have been studying the effects of Mtb chemotherapy on the host microbiota and the possible influence of such changes on immunological responsiveness to infection and the pharmacokinetics of the TB drugs themselves. Our work had indicated that the standard TB drugs (isoniazid, pyrazinamide and rifampicin) given to patients as a cocktail induce major changes in the distribution of gut bacterial species in mice as determined by 16s RNA sequencing as early as 2 wks post-treatment. This year Sivaranjani Namasivayam extended these studies and showed that the dysbiosis induced by TB chemotherapy persists for at least 3 months after the cessation of drug administration. Careful analysis of the sequence data revealed prominent changes in the class Clostridia and in particular the family Lachnospiraceae in the animals receiving chemotherapy. She also performed experiments administering the individual drugs in the cocktail and showed that rifampicin alone or in combination with pyrazinamide accounted for most of the changes in gut bacterial distribution. Finally, Dr. Namasivayam initiated new experiments aimed at amplifying and analyzing commensal bacteria sequences in the lungs of TB infected mice. The NOD receptors are cytoplasmic pattern recognition receptors that recognize bacterial cell wall peptidoglycans. As indicated in an accompanying report, we have shown that NOD 1 signalling influences the generation of common lymphoid progenitor cells in the bone marrow. In related experiments Chiaki Iwamura asked whether NOD stimulation might regulate the function of non-hematopoietic cells at the same site. In particular he focused on mesenchymal stromal cells that are known to produce a wide variety of different cytokines many of which also have a role in steady-state homeostasis. He demonstrated that NOD1, but not NOD2 ligand stimulation of bone marrow mesenchymal stromal cells in vitro induced expression of multiple hematopoietic cytokines (IL-7, Flt3L, SCF, ThPO and IL-6). Importantly, in vivo administration of NOD1 ligand to germ-free mice that have a decreased pool of hematopoietic precursors resulted in restoration of the numbers of bone marrow hematopoietic stem cells and precursors as well as serum concentrations of IL-7, Flt3L, SCF and ThPO to the levels displayed by specific pathogen free control animals. Based on these findings we propose that NOD1 signaling in mesenchymal stromal cells represents as an important pathway underlying the requirement for microbiota in the maintenance of steady-state hematopoiesis. The latter function is distinct from that previously shown to be triggered by lipopolysaccharide in both its broad effects on multiple progenitors and specific targeting of mesenchymal stromal cells as cytokine producing intermediates.